Methods for inhibiting the expression of a target gene in cells, tissues, or individuals include an approach in which double-stranded RNA is introduced into the cells, tissues, or individuals. By this introduction of double-stranded RNA, mRNA having homology to the sequence is degraded such that the expression of the target gene is inhibited. This effect is called “RNA interference” or “RNAi”. RNA interference was originally reported in C. elegans (see e.g., Non Patent Reference 1) and then also reported in plants (see e.g., Non Patent Reference 2).
Double-stranded RNA consisting of 21-nucleotide sense and antisense strands having a 2-nucleotide overhang at the 3′-end (small interfering RNA: siRNA) has been reported to have an RNA interference effect in cultured cells of vertebrates (see e.g., Non Patent Reference 3). siRNA is considered to be useful for the identification of gene functions, screening of cell strains suitable for useful substance production, regulation of genes involved in disease, etc., but, however, it is characteristically degraded easily by RNase (see e.g., Non Patent Reference 4).
A double-stranded polynucleotide having nucleotide units of alternately combined DNAs and 2′-OMeRNAs, instead of RNAs constituting siRNA, has been reported as being a double-stranded polynucleotide that is resistant to RNase and has an RNA interference effect (see Patent Reference 1).
Some reports have been made on the modification of the 5′-ends of sense and antisense strands in siRNA. It has been reported that siRNA having a 6-aminohexyl phosphate group at the 5′-end of the sense or antisense strand has inhibitory activity against the expression of target mRNA (see Non Patent Reference 5). On the other hand, it has been reported that siRNA having this 6-aminohexyl phosphate group at the 5′-end of the antisense strand has no inhibitory activity against the expression of target mRNA (see e.g., Non Patent Reference 6). It has also been reported that siRNA having a 3-aminopropyl phosphate group at the 5′-end of the sense strand has inhibitory activity against the expression of target mRNA, whereas siRNA having a 3-aminopropyl phosphate group at the 5′-end of the antisense strand has no inhibitory activity against the expression of target mRNA (see e.g., Non Patent Reference 7). It has been reported that inhibitory activity against the expression of target mRNA is observably lower in siRNA having the 6-aminohexyl phosphate group or the 3-aminopropyl phosphate group at the 5′-end of the antisense strand than in unmodified siRNA but it is not completely lost. (see e.g., Non Patent Reference 8).
It has been reported that siRNA having fluorescein at the 5′-end of the sense or antisense strand also has inhibitory activity against the expression of target mRNA (see e.g., Non Patent Reference 9). It has been reported that of siRNAs having a steroid or lipid structure at the 5′-end of the sense or antisense strand, siRNA having a steroid or lipid structure at the 5′-end of the sense strand has inhibitory activity against the expression of target mRNA (see e.g., Non Patent Reference 8). It has been reported that when siRNA has an ortho-nitrobenzyl derivative, which can be eliminated by UV irradiation, at the 5′-end of the antisense strand, its inhibitory activity against the expression of target mRNA can be controlled by using UV irradiation (see e.g., Non Patent Reference 10).
siRNA in which the 3′-end of the sense strand and the 5′-end of the antisense strand are linked via a loop consisting of approximately 4 nucleotide units forms a single-stranded polynucleotide called short hairpin RNA (shRNA). shRNA having a 19-bp stem moiety has been shown to have lower activity than that of a 19-bp siRNA having the same nucleotide sequence thereas (see e.g., Non Patent Reference 9). Although shRNA comprising a 19-bp stem and a loop having two nucleotides replaced with a non-nucleotide linker such as propyl phosphate units was synthesized and examined for its inhibitory activity against the expression of target mRNA, no improvement in activity was observed, compared with unmodified shRNA (see e.g., Non Patent Reference 9). An example using an ortho-nitrobenzyl derivative has been reported as siRNA in which the 3′-end of the sense strand and the 5′-end of the antisense strand are linked via a non-nucleotide linker (see e.g., Non Patent Reference 8). This 19-bp siRNA whose sense and antisense strands are linked via the ortho-nitrobenzyl derivative has lower inhibitory activity against the expression of target mRNA than that of unmodified siRNA. In addition, cultured cells transfected with this siRNA were irradiated with UV for 10 minutes and examined for the inhibitory activity of the siRNA against the expression of target mRNA. As a result, the inhibitory activity against the expression of target mRNA was lower than that of unmodified siRNA. A single-stranded polynucleotide having a structure in which the 5′-end of the antisense strand and the 3′-end of the sense strand are linked via a phenyl group-containing linker to form a phosphodiester structure at each of these ends is not yet known.
X-ray analysis of a complex of an antisense strand with Argonaute protein (Ago) known to participate in RNAi activity has showed that the 5′-terminal phosphate group of the antisense strand and its neighboring nucleotides are strongly bound to the PIWI domain of Ago (see e.g., Non Patent Reference 11). It has been reported that upon introduction of chemically synthesized siRNA into cells, both sense and antisense strands are 5′-terminally phosphorylated (see e.g., Non Patent Reference 12). In human cells, RNA kinase hC1p1 has been reported to be responsible for the 5′-phosphorylation of siRNA (see e.g., Non Patent Reference 13). When 5′-terminally phosphorylated siRNA and siRNA having an unphosphorylated 5′-end were separately introduced into cells and their RNAi activities were compared, no difference in activity was seen therebetween, indicating that the siRNA having an unphosphorylated 5′-end is easily subject to phosphorylation in cells (see e.g., Non Patent Reference 9).
In the case of using the shRNA in which the 3′-end of the sense strand and the 5′-end of the antisense strand are linked via a loop, this shRNA is intracellularly cleaved by the Dicer protein or an endonuclease to form an antisense strand having a 5′-terminal phosphate group (see e.g., Non Patent Reference 9). The shRNA comprising a 19-bp stem and a loop having two nucleotides replaced with propyl phosphate units cannot be expected to undergo intracellular Dicer or endonuclease cleavage, because the propyl phosphate units are resistant to nuclease (see e.g., Non Patent Reference 9). Alternatively, the shRNA comprising a 19-bp stem and an ortho-nitrobenzyl derivative loop can be expected to form an antisense strand having a 5′-terminal phosphate group by UV irradiation. Such UV irradiation, however, is difficult to apply to living bodies due to possible adverse reactions and due to the difficulty of applying UV irradiation inside a living body (see e.g., Non Patent Reference 8). A single-stranded polynucleotide comprising a 19-bp or less stem and a loop having a non-nucleotide linker alone, which is intracellularly cleaved by Dicer or endonuclease without UV irradiation to form an antisense strand having a 5′-terminal phosphate group, is not yet known.
The present inventors have conducted diligent studies to obtain a polynucleotide that has an RNA interference effect and/or a gene expression inhibitory effect, and have consequently completed the present invention by finding a single-stranded polynucleotide having an RNA interference effect and/or a gene expression inhibitory effect, which is derived from a double-stranded polynucleotide comprising a sense strand polynucleotide corresponding to a target gene, and an antisense strand polynucleotide having a nucleotide sequence complementary to the sense strand polynucleotide, and has a structure in which the 5′-end of the antisense strand and the 3′-end of the sense strand are linked via a phenyl group-containing linker to form a phosphodiester structure at each of these ends.